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Mass administration of macrocyclic lactones targets the transmission of the causative agents of lymphatic filariasis to their insect vectors by rapidly clearing microfilariae (Mf) from the circulation. It has been proposed that the anti-filarial action of these drugs may be mediated through the host immune system. We recently developed an in vitro assay for monitoring the attachment to and killing of B. malayi Mf by human neutrophils (PMNs) and monocytes (PBMCs), however, the levels of both cell to worm attachment and leukocyte mediated Mf killing varied greatly between individual experiments. To determine whether differences in an individual's immune cells or the Mf themselves might account for the variability in survival, PMNs and PBMCs were isolated from 12 donors every week for 4 weeks and the cells used for survival assays with a different batch of Mf, thereby keeping donors constant but varying the Mf sample. Results from these experiments indicate that, overall, killing is Mf-rather than donor-dependent. To assess whether ivermectin (IVM) or diethylcarbamazine (DEC) increase killing, Mf were incubated either alone or with immune cells in the presence of IVM or DEC. Neither drug induced a significant difference in the survival of Mf whether cultured with or without cells, with the exception of DEC at 2 h post incubation. In addition, human PBMCs and PMNs were incubated with IVM or DEC for 1 h or 16 h prior to RNA extraction and Illumina sequencing. Although donor-to-donor variation may mask subtle differences in gene expression, principle component analysis of the RNASeq data indicates that there is no significant change in the expression of any genes from the treated cells versus controls. Together these data suggest that IVM and DEC have little direct effect on immune cells involved in the rapid clearance of Mf from the circulation., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Calreticulin (CRT), a highly conserved ubiquitous eukaryotic protein with a molecular mass of 46 kDa, containing three domains (N, P, and C) is involved in promoting prolonged parasite-host relations. Brugia malayi Calreticulin (BmCRT) is involved in the establishment of parasite infection by suppression of C1q-mediated host immune response. Calcium plays important role in this immunomodulatory mechanism of BmCRT. In the present study binding of calcium with BmCRT region involved in this interaction was investigated and correlated with the accompanying changes in fluorescence, circular dichroism (CD) and UV absorption. The results obtained clearly indicated that BmCRT is a calcium binding protein and contains types two of Ca 2+ binding sites, one high affinity Ca 2+ binding site at P domain and another low affinity Ca 2+ binding site at C domain. Zinc also binds to additional sites that do not have appreciable affinity for the calcium. These studies have provided new knowledge that allows us to describe how the structure of BmCRT responds to interactions with calcium and zinc which is different from human CRT and also discuss how this mechanism help to complex formation with host C1q., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses., Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays., Results: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05)., Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Coinfections are common in natural populations and the outcome of their interactions depends on the immune responses of the host elicited by the parasites. Earlier we showed that immunization with BmAFII (Sephadex G-200 eluted) fraction of human lymphatic filaria Brugia malayi inhibited progression of Leishmania donovani infection in golden hamsters. In the present study we identified cross reactive molecules of B. malayi, and investigated their effect on L. donovani infection and associated immune responses in the host. The sequence alignment and sharing of linear T- and B-cell epitopes in protein molecules of B. malayi and L. donovani counterparts were studied in silico. Hamsters were immunized with robustly cross reactive SDS-PAGE resolved fractions F6 (54.2-67.8kDa) and F9 (41.3-45.0kDa) of B. malayi and subsequently inoculated with amastigotes of L. donovani intracardially. F6 inhibited (∼72%) L. donovani infection and upregulated Th1 cytokine expression, lymphoproliferation, IgG2, IgG2/3 levels and NO production, and downregulated Th2 cytokine expression. Sequences in HSP60 and EF-2 of F6 and L. donovani counterparts were conserved and B- and T-cell epitopes in the proteins shared antigenic regions. In conclusion, leishmania-cross reactive molecules of filarial parasite considerably inhibited leishmanial infection via Th1-mediated immune responses and NO production. Common B- and T-cell epitope regions in HSP60 and EF-2 of the parasites might have contributed to the inhibitory effect on the L. donovani infection. Thus, leishmania-cross reactive filarial parasite molecules may help in designing prophylactic(s) against L. donovani., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lymphatic filariasis (LF), a morbid vector-borne parasitic infection affects millions in tropical areas. Complete eradication can only be achieved by the development of a potent vaccine. Among the various filarial antigens that have been characterized, antigens Brugia malayi thioredoxin (TRX) and abundant larval transcript (ALT) have produced recognizable level of protection in Jirds, thereby evidenced to be good vaccine candidates. In this study an attempt was made to enhance their immunoprophylactic activity by encapsulating them in natural polysaccharide chitosan forming nanospheres (CN). High encapsulation efficiency for TRX (93%) in CN (TCN) and ALT-2 (90%) in CN (ACN) was achieved. Morphological studies confirmed the spherical and uniform distribution of nanospheres to be 220 nm. The electrostatic interaction between chitosan and the antigens were confirmed using differential scanning calorimetry and FT-IR. The study revealed the immunostimulatory property of chitosan providing enhanced level of proliferation for encapsulated antigens in peripheral blood mononuclear cells from endemic normal personals, at low concentration (TCN mean stimulation index (SI)=4.23±0.15 and ACN (SI)=4.05±0.33) compared to stimulation obtained by antigens alone. Hence, our study demonstrated that natural macromolecule derived CN can be used as efficacious immunostimulatory vaccine carter for LF thereby diminishing pathological sequel., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Alternative macrophage activation is largely defined by IL-4Rα stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-α or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-α or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine candidate because it is present in all the major life stages of parasite, but is absent in mammals. We have previously cloned, purified and biochemically characterized Bm-TPP. In the present study, we investigated the cross-reactivity of recombinant Bm-TPP (r-Bm-TPP) with the sera of human bancroftian patients belonging to different disease categories. In silico study using bioinformatics tool demonstrated that Bm-TPP is highly immunogenic in nature. BALB/c mice administered with r-Bm-TPP alone or in combination with Freund's complete adjuvant (FCA) generated a strong IgG response. Further investigations on the antibody isotypes showed generation of a mixed T helper cell response which was marginally biased towards Th1 phenotype. r-Bm-TPP with or without adjuvant lead to significantly increased accumulation of CD4+ and CD8+ T cells in the spleen of infected mice and increased the activation of peritoneal macrophages. Additionally, r-Bm-TPP enhanced the production of both proinflammatory (IL-2, IFN-γ) and anti-inflammatory (IL-4, IL-10) cytokines and mice immunized with r-Bm-TPP alone or in combination with FCA showed 54.5% and 67% protection respectively against B. malayi infective larvae challenge. Taken together, our findings suggest that Bm-TPP is protective in nature and might be a potential candidate for development of vaccine against lymphatic filarial infections., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Tropical pulmonary eosinophilia is prevalent in the tropical and subtropical regions of the world. It is an occult form of human filariasis and results from an exaggerated immune response to filarial parasites Wuchereria bancrofti and Brugia malayi. Tuberculosis is prevalent in our country and may mimic almost any pulmonary disease on chest skiagram. Here we describe a patient with acute chest symptoms and micro-nodular opacity over chest roentogenogram, diagnosed as miliary tuberculosis and treated accordingly. Actually he was suffering from tropical pulmonary eosinophilia and showed response to combined diethylcarbamazine and corticosteroid therapy. This case serves as a reminder that tropical pulmonary eosinophilia may be wrongly diagnosed as miliary tuberculosis if one rely solely on a chest X-ray with micronodular opacities. We also stress on early diagnosis and treatment of this condition to avoid unfavorable outcomes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice., Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA., Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-γ in serums from the immunized mice were significantly higher than that of the control (P value <0.05)., Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Filarial parasites survive by inducing tolerance in host but the antigens and mechanisms involved are not clear. Recently we found that BmAFI, a Sephadex G-200 eluted fraction of Brugia malayi adult worm extract, stimulates IL-10 release from THP-1 cells. In the present study, we determined the SDS-PAGE profile of BmAFI and infective 3rd stage larva (L3), investigated the effect of pre-sensitization of host with BmAFI on the survival and development of L3 in the non-permissive peritoneal cavity (p.c.) of the permissive host Mastomys coucha and in the p.c. of non-permissive Swiss mice, and studied immunological correlates for the observed effects. The parasite development and burden in p.c., was determined in sensitized infected M. coucha and Swiss mice and the release of TGF-β, IL-4, IL-10, IL-13, IFN-γ and NO, cellular proliferative response to Con A and BmAFI and levels of IgG subclasses and IgE were determined in sensitized infected M. coucha. Cellular proliferative response to Con A and BmAFI, mRNA expression of GATA-3, CTLA-4 and T-bet were determined in sensitized Swiss mice. In addition, the parasitological parameter was also studied in BmAFI-sensitized M. coucha exposed to the infection by standard subcutaneous (s.c.) route to assess whether sensitization enhances the intensity of infection. BmAFI-sensitization permitted survival of L3 and their development to adult stage by day 60 p.i. in the p.c. of M. coucha; in non-sensitized animals L3 could molt to L4 only and no parasite could be recovered beyond day 30 p.i. In M. coucha that received infection by s.c. route, pre-sensitization with BmAFI enhanced the microfilaraemia and adult worm recovery. In sensitized Swiss mice L3 could successfully molt to L4 in p.c. with improved recovery of parasite. BmAFI sensitization upregulated TGF-β and IL-10 release, IgG1 and IgG2b levels, GATA-3 and CTLA-4 mRNA expression, suppressed the cellular proliferative response and downregulated Con A stimulated response, IgE, IL-13, IFN-γ and NO responses. Immunoblot analysis showed that the BmAFI antiserum also strongly reacts with some L3 molecules. The results show, for the first time, that sensitization with the anti-inflammatory BmAFI which shares some of its molecules with those in L3, facilitates parasite survival in the non-permissive p.c. of the permissive host M. coucha, render a non-permissive Swiss mouse partially permissive to infection and enhances parasite load in M. coucha receiving the infection through permissive s.c. route by evoking a modified Th2 type of response and anti-inflammatory milieu. In conclusion, the findings suggest that the anti-inflammatory BmAFI fraction facilitates survival of B. malayi infection even in non-permissive environment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Wolbachia, an endosymbiont present in filarial nematodes, have been implicated in a variety of roles, including the worm development and survival. Elucidation of the role of Wolbachia in filarial nematode biology and pathogenesis has become the focus of many studies and its contribution to parasite survival or immune response is still unclear. Recombinant Wolbachia HSP60 decreases T cell activation and lymphoproliferation in filarial infected people compared to endemic controls as observed by the assessment of T cell activation markers and cytokine responses in the peripheral blood mononuclear cells. Reduced T cell activation may be linked to T regulatory cell activity since it is associated with increased expression of CTLA4 and CD25 on CD4(+) T cells in filarial infected group upon stimulation with recombinant Wolbachia HSP60. In addition, elevated interleukin-10 and TGF-β cytokines corroborate the reduced CD4(+) T cell activation and interferon-γ observed upon recombinant Wolbachia HSP60 stimulation in filarial patients. Hence, these findings indicate that Wolbachia HSP60 may also contribute to the immune modulation seen in filarial patients., (Copyright © 2011 Institut Pasteur. All rights reserved.)

Antibody tests are useful for mapping the distribution of lymphatic filariasis (LF) in countries and regions and for monitoring progress in elimination programs based on mass drug administration (MDA). Prior antibody tests have suffered from poor sensitivity and/or specificity or from a lack of standardization. We conducted a multicenter evaluation of a new commercial ELISA that detects IgG4 antibodies to the recombinant filarial antigen Bm14. Four laboratories tested a shared panel of coded serum or plasma samples that included 55 samples from people with microfilaremic Wuchereria bancrofti or Brugia infections and 26 control samples. Qualitative results were identical in all four test sites. In addition, each laboratory tested samples from their own serum banks. The test detected antibodies in 32 of 36 samples (91%) from people with Brugian filariasis and in 96 of 98 samples (98%) from people with Bancroftian filariasis. Specificity testing showed that many serum or plasma samples from patients with other filarial infections such as onchocerciasis had positive antibody tests. Specificity was otherwise excellent, although 3 of 30 samples from patients with ascariasis and 4 of 51 with strongyloidiasis had positive antibody tests; it is likely that some or all of these people had previously lived in filariasis-endemic areas. Antibody test results obtained with eluates from blood dried on filter paper were similar to those obtained with plasma tested at the same dilution. This test may be helpful for diagnosing LF in patients with clinical signs of filariasis. It may also be a useful tool for use in LF endemic countries to monitor the progress of filariasis elimination programs and for post-MDA surveillance., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Filariasis remains a health problem in tropical countries. Identification of immunogens from its causative organism would lead to development of a better diagnostic test, as well as vaccine discovery to effectively prevent this disease. We applied immunoproteomics to define potential immunogens of adult Brugia malayi that were recognized by IgM, IgG1 and IgG4 in sera of patients with four distinct clinical spectra of filariasis, including endemic asymptomatic, lymphangitis, elephantiasis and microfilaremia (n=5/group). Sera of healthy individuals (n=5) from non-endemic area served as the negative control. Brugian proteins were resolved by 2-DE and subjected to 2-D Western blot analysis probed with these sera. A total of 30 immunoreactive proteins recognized by IgM, IgG1 and IgG4 in sera from all four filarial groups were identified by Q-TOF MS and MS/MS analyses. Interestingly, only three immunogens were recognized by IgM in lymphangitis, elephantiasis and microfilaremia, but not in endemic asymptomatic group. IgG1 recognized 20 immunogens in endemic asymptomatic, lymphangitis and microfilaremia (mostly in endemic asymptomatic group), but not in elephantiasis, whereas IgG4 recognized 28 immunogens in all four filarial groups (mostly in microfilaremia). This large data set is an important resource for further development of a new diagnostic test and/or vaccine for filariasis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although the antioxidant thioredoxin peroxidase (TPX) is a putative target exploited in vaccine studies of lymphatic filariasis, the high sequence homology with host peroxiredoxins remains a great concern. The emergence of immunomics offers a powerful tool for novel vaccine design. Further, due to the cellular hypo-response in filariasis, analysis of T epitope repertoire becomes imperative in disease control. Here, we report the cellular responses of filarial TPX-1 and the identification of T epitope (29-43) in the host non-homologous region. The strong proliferative responses induced by the peptide mimetic in mice splenocytes and human PBMC's prove the existence of T epitope recognized in endemic population., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

We earlier reported a sizeable protection conferred by 'mitochondria rich' (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of approximately 34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-alpha, IFN-gamma and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that approximately 34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.

Although DNA vaccines have several advantages over conventional vaccines, antibody production and protection are often not adequate, particularly in single plasmid vaccine formulation. In the present study we evaluated the efficacy of a cocktail vaccine based on plasmids encoding larval (L3) stage-specific Brugia malayi abundant larval transcript (BmALT-2) and antioxidant detoxification enzyme B. malayi thioredoxin peroxidase (BmTPX) to induce antibodies, protective efficacy and cell-mediated immune response in mice. Mice immunized with cocktail DNA vaccines containing the pVAX ALT-2+TPX developed higher titers of anti-BmALT-2+TPX (1:5000) antibodies, compared to the mice immunized with single DNA vaccine of pVAX ALT-2 or pVAX TPX (1:2000). Correlating with this, the mice administered with cocktail vaccine induced up to 78% of cytotoxicity against B. malayi mf. This cytotoxicity was high compared to 34% induced by the pVAX-ALT2 or 37% by pVAX-TPX. Moreover, cocktail vaccination of mice resulted in significantly higher level of cellular proliferative response associated with raised levels of IFN-gamma that skewed towards Th1 type of response compared to vaccination using either of the components. Taken together, these data suggest that the combination of two or more antigens maybe an effective vaccine development strategy to improve protection and immunogenicity against human lymphatic filariasis.

The influence of live Brugia malayi parasites and a Sephadex G-200 fraction of the adult parasite extract (BmAFII) on the progression of Leishmania donovani infection was studied. Inbred hamsters were first infected with B. malayi infective 3rd stage larvae (L3), adult worms or microfilariae (mf), and then with L. donovani amastigotes (Ld), or vice versa or received both the infections simultaneously; a group of animals were first immunized with BmAFII and then infected with Ld. L. donovani parasite burden was determined between 17 and 19 days post amastigote challenge (p.a.c.) and, in case of immunized animals, between 32 and 35 days p.a.c also. Nitric oxide (NO) release from peritoneal macrophages and cellular proliferative responses of lymphnode cells were assessed in BmAFII-immunized animals given leishmania infection or no infection. Leishmanial parasite burden was significantly reduced in animals exposed to filarial L3 before amastigote inoculation and in animals given filarial adult worms after or together with amastigotes. Prior immunization of leishmania-infected animals with BmAFII also reduced the leishmanial parasite burden (17-19 days p.a.c.: >90%; 32-35 days p.a.c.: 60%). These animals showed upregulation of NO release and cellular proliferative responses to promastigote antigen or BmAFII stimulation in vitro. The findings show, for the first time, that B. malayi L3/adult worms or immunization with BmAFII inhibits progression of L. donovani infection in hamsters and this is associated with upregulation of NO and lymphocyte proliferative responses indicating that Th1 response might be responsible for this.

The Brugia malayi filarial antigens recognised preferentially by sera from an endemic normal population are considered to be potential vaccine candidates. By immunoscreening the cDNA library of the infective L3 stage of B. malayi with pooled endemic normal sera, a cDNA clone Bm-SL3 was identified. Analysis of sera from different patient groups with the rBm-SL3 protein showed it to be highly reactive with endemic normal sera compared to its reactivity with microfilaraemic and non-endemic normal sera. The immunoprotective efficacy of the rBm-SL3 antigen against B. malayi filarial infection was evaluated in susceptible host jirds (gerbils) (Meriones unguiculatus). Jirds immunised with the rBm-SL3 antigen showed 68% cytotoxicity against microfilariae and 67-69% cytotoxicity against infective larvae in in-vitro antibody-dependent cellular cytotoxicity assays and in-situ micropore chamber methods. Analysis of IgG subclasses against Bm-SL3 revealed a significant increase in IgG1 and IgG2 antibodies in endemic normal sera compared with other groups. Lymphocyte proliferation to Bm-SL3 was significantly higher in the endemic normal group compared with that in clinical and microfilarial carriers (p < 0.001). Significantly enhanced levels of IFN-gamma in the culture supernatant of peripheral blood mononuclear cells of endemic normal sera after stimulation with Bm-SL3 suggest that the cellular response in this group may have a Th1 bias.

A 43 kiloDaltan (kDa) antigen fraction (CFA2-6) isolated from microfilaraemic plasma of bancroftian filarial patients showed selective reactivity with sera samples collected from endemic normals. Antibodies raised against this antigen showed a strong reactivity with the surface of Brugia malayi infective larvae as well as microfilariae. Similar antigenic determinants were detected in the parasite extracts, but not in the excretory-secretory products. Further analysis was done on the immunoprophylactic potential of CFA2-6 in inducing immunity against Brugia malayi in Meriones unguiculatus (jird) in vivo. A strong protective response of approximately 84% was observed against the development of the filarial parasite in the jirds immunized with CFA2-6. The immunized jirds also showed a significant clearance (87%) of microfilariae inoculated intravenously. Approximately 65% of infective larvae failed to survive in jirds transferred with anti-CFA2-6 serum compared to the jirds transferred with sera from the control jirds. Passive transfer of anti-CFA2-6 antibody to the jirds followed by intravenous inoculation of microfilariae resulted in the reduction of 77% of circulating microfilariae. This study suggests that the 43-kDa CFA2-6 could stimulate a strong protective immune response against infective larvae and microfilariae in experimental animals.

Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individuals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti-filarial IgG4 antibodies, 200 sera from healthy city-dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10-15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solution, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in between each incubation step. Luminol chemiluminescence detection was then used to develop the blots. An antigenic band with the MW of approximately 37 kDa was found to be consistently present in the Western blots of all microfilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated patients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individuals, and city dwellers. Therefore the B. malayi antigen of with the MW of approximately 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application., (Copyright 1998 Academic Press.)

T and B lymphocyte-deficient mice homozygous for the severe combined immunodeficiency (SCID) mutation can be immunologically engrafted with human lymphocytes. However, low levels of human peripheral blood mononuclear cell engraftment are commonly observed, impeding full use of this model. We now demonstrate that strain background in mice homozygous for the scid mutation is a strong determinant of levels of human lymphocyte engraftment. NOD/LtSz-scid/scid mice support higher levels of engraftment of both human spleen and peripheral blood mononuclear cells than do C.B-17-scid/scid mice. We observed, using human spleen cell injected scid mice, 1), high levels of engraftment of the host peripheral lymphoid tissues with human CD45+ (leukocytes), CD3+ (T cells), CD4+ (helper/inducer), and CD8+ (suppressor/cytotoxic) lymphoid cells for up to 24 weeks in NOD/LtSz-scid/scid mice; 2), migration of high numbers of human lymphocytes to peripheral lymphoid and nonlymphoid organs in NOD/LtSz-scid/scid, but not in C.B-17-scid/scid mice; 3), higher levels of serum immunoglobulin of human origin in NOD/LtSz-scid/scid mice than in C.B-17-scid/scid mice; 4), histological lesions characteristic of human anti-mouse xenoreactivity in NOD/LtSz-scid/scid mice; and 5), human origin antibodies against filarial antigens after engraftment with native human spleen cells. The use of NOD/LtSz-scid/scid mice as recipients to achieve significantly enhanced human lymphopoietic cell engraftment will now enable human immunity to be more easily studied in animal models.

We have used the severe combined immunodeficient C.B-17-scid/scid mouse to investigate the influences of maternal immune status and parasite burden on the susceptibility (or resistance) of offspring to infection with the human filarial parasite, Brugia malayi. C.B-17-scid/scid mice are permissive for infection while immunocompetent C.B-17(-)+/+ mice are uniformly resistant. Reciprocal matings of C.B-17-scid/scid and C.B-17(-)+/+ mice were performed. The C.B-17-scid/scid females were either naive or infected with Brugia malayi. The resulting immunocompetent C.B-17-scid/+ and C.B-17(-)+/scid progeny were challenged at weaning with an intraperitoneal injection of Brugia malayi third stage larvae known to produce patent infection in > 95% of C.B-17-scid/scid mice. We observed that 40.0%l (34/85) of the immunocompetent offspring of C.B-17-scid/scid females x C.B-17(-)+/+ males were permissive for the growth and development of Brugia malayi larvae to adults. No difference was observed in susceptibility to infection between the progeny of infected or uninfected C.B-17-scid/scid mothers mated with C.B-17(-)+/+ fathers, arguing against acquired immunological tolerance to the parasite in the former. In marked contrast, only 4.8% (2/42) of the heterozygous progeny of wild type C.B-17(-)+/+ females mated with C.B-17-scid/scid males were permissive. These observations document conversion of a 'resistant' phenotype to a 'susceptible' phenotype by manipulation of maternal immune status and provide clear evidence of maternal influence on offspring susceptibility to infection with Brugia malayi.

Sera from fifty subjects with different presentations of Brugian filariasis and from common soil-transmitted helminth infections were tested for specific anti-filarial IgG and its subclasses. Anti-filarial IgG, IgG1 and IgG3 showed cross-reactivities with soil-transmitted helminthic infections and no significant differences in optical densities among the various groups of filarial patients. In comparison with other groups of subjects, IgG4-ELISA of sera from microfilaraemic patients and some previously microfilaraemic patients showed a significant increase in optical density readings, while IgG2-ELISA showed elevated optical density readings in sera of patients with chronic elephantiasis. Therefore IgG2-ELISA is potentially useful in the diagnosis of brugian chronic elephantiasis while IgG4-ELISA may be beneficial for follow-up diagnosis of treated microfilaraemic patients.